Effect of Gravity Changes on the Cyanobacterium Synechocystis sp. PCC 6803

The impact of hypergravity and simulated weightlessness were studied to check whether cyanobacteria perceive changes of gravity as stress. Hypergravity generated by a low-speed centrifuge increased slightly the overall activity of dehydrogenases, but the increase was the same for 90 g and 180 g. The protein pattern did not show qualitative alterations during hypergravity treatment up to 180 g. Cells of Synechocystis PCC 6803 subjected to common stressors like salt, heat, and light clearly accumulated at least four general stress proteins (25, 31, 34, and 63 kDa, respectively). Three of these proteins could also be detected after hypergravity, but in such small amounts that their occurrence could only be taken as a weak indication of stress. Low-molecular-weight stress metabolites were not synthesized in response to hypergravity, indicating that this gravity change was unable to activate the osmotic signal transduction chain. Gravity-dependent alterations were observed only during simulated weightlessness (generated by a fast-rotating clinostat). The glutamate/glutamine ratio was significantly shifted toward a higher glutamine portion. Altogether, the results may indicate that moderate changes of gravity were hardly, if ever, sensed as stress by cyanobacteria. Received: 20 May 1997 / Accepted: 25 June 1997     

Temperature-Induced Changes in the Fatty Acid Composition of the Cyanobacterium, Synechocystis PCC6803

Changes in glycerolipid and fatty acid composition with a change in growth temperature were studied in the cyanobacterium, Synechocystis PCC6803. Under isothermal growth conditions, temperature did not significantly affect the composition of the various classes of lipids, but a decrease in temperature altered the degree of unsaturation of C₁₈ acids at the sn-1 position, but not that of C₁₆ acids at the sn-2 position of the glycerol moiety in each class of lipids. When the growth temperature was shifted from 38°C to 22°C, the desaturation of C₁₈ acids, but not that of C₁₆ acids, was stimulated. The desaturation of fatty acids occurred only in the light and was inhibited by chloramphenicol, rifampicin and 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, but not by cerulenin, an inhibitor for fatty acid synthesis. These findings suggest that desaturase activities are induced after a shift from a higher to a lower temperature, and that the desaturation of fatty acids is connected with the reactions involved in photosynthetic electron transport.     

Genomic Responses to Arsenic in the Cyanobacterium Synechocystis sp. PCC 6803

Arsenic is a ubiquitous contaminant and a toxic metalloid which presents two main redox states in nature: arsenite [As^III] and arsenate [As^V]. Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by the arsBHC operon and two additional arsenate reductases encoded by the arsI1 and arsI2 genes. Here we describe the genome-wide responses to the presence of arsenate and arsenite in wild type and mutants in the arsenic resistance system. Both forms of arsenic produced similar responses in the wild type strain, including induction of several stress related genes and repression of energy generation processes. These responses were transient in the wild type strain but maintained in time in an arsB mutant strain, which lacks the arsenite transporter. In contrast, the responses observed in a strain lacking all arsenate reductases were somewhat different and included lower induction of genes involved in metal homeostasis and Fe-S cluster biogenesis, suggesting that these two processes are targeted by arsenite in the wild type strain. Finally, analysis of the arsR mutant strain revealed that ArsR seems to only control 5 genes in the genome. Furthermore, the arsR mutant strain exhibited hypersentivity to nickel, copper and cadmium and this phenotype was suppressed by mutation in arsB but not in arsC gene suggesting that overexpression of arsB is detrimental in the presence of these metals in the media.     

Complete Genome Structure of the Unicellular Cyanobacterium Synechocystis sp. PCC6803

Cyanobacteria are photoautotrophic organisms capable of oxygen-producingphotosynthesis similar to that in eukaryotic algae and plants,and because of this, they have been used as model organismsfor the study of the mechanism and regulation of oxygen-producingphotosynthesis. To understand the entire genetic system in cyanobacteria,the nucleotide sequence of the entire genome of the unicellularcyanobacterium Synechocystis sp. PCC6803 has been determined.The total length of the circular genome is 3,573,470 bp, witha GC content of 47.7%. A total of 3,168 potential protein codinggenes were assigned. Of these, 145 (4.6%) were identical toreported genes, and 1,259 (39.6%) and 342 (10.8%) showed similarityto reported and hypothetical genes, respectively. The remaining1,422 (45.0%) showed no apparent similarity to any genes registeredin the databases. Classification of the genes by their biologicalfunction and comparison of the gene complement with those ofother organisms have revealed a variety of features of the geneticinformation characteristic of a photoautotrophic organism. Thesequence data, as well as other information on the Synechocystisgenome, is presented in CyanoBase on WWW [http://www.kazusa.or.jp/cyano/]. (Received July 24, 1997; Accepted September 17, 1997)     

Genomic Structure of the Cyanobacterium Synechocystis sp. PCC 6803 Strain GT-S

Synechocystis sp. PCC 6803 is the most popular cyanobacterial strain, serving as a standard in the research fields of photosynthesis, stress response, metabolism and so on. A glucose-tolerant (GT) derivative of this strain was used for genome sequencing at Kazusa DNA Research Institute in 1996, which established a hallmark in the study of cyanobacteria. However, apparent differences in sequences deviating from the database have been noticed among different strain stocks. For this reason, we analysed the genomic sequence of another GT strain (GT-S) by 454 and partial Sanger sequencing. We found 22 putative single nucleotide polymorphisms (SNPs) in comparison to the published sequence of the Kazusa strain. However, Sanger sequencing of 36 direct PCR products of the Kazusa strains stored in small aliquots resulted in their identity with the GT-S sequence at 21 of the 22 sites, excluding the possibility of their being SNPs. In addition, we were able to combine five split open reading frames present in the database sequence, and to remove the C-terminus of an ORF. Aside from these, two of the Insertion Sequence elements were not present in the GT-S strain. We have thus become able to provide an accurate genomic sequence of Synechocystis sp. PCC 6803 for future studies on this important cyanobacterial strain.     

Type 2 NADH Dehydrogenases in the Cyanobacterium Synechocystis sp. Strain PCC 6803 Are Involved in Regulation Rather Than Respiration

Analysis of the genome of Synechocystis sp. strain PCC 6803 reveals three open reading frames (slr0851, slr1743, and sll1484) that may code for type 2 NAD(P)H dehydrogenases (NDH-2). The sequence similarity between the translated open reading frames and NDH-2s from other organisms is low, generally not exceeding 30% identity. However, NAD(P)H and flavin adenine dinucleotide binding motifs are conserved in all three putative NDH-2s in Synechocystis sp. strain PCC 6803. The three open reading frames were cloned, and deletion constructs were made for each. An expression construct containing one of the three open reading frames, slr1743, was able to functionally complement an Escherichia coli mutant lacking both NDH-1s and NDH-2s. Therefore, slr0851, slr1743, and sll1484 have been designated ndbA, ndbB, and ndbC, respectively. Strains that lacked one or more of the ndb genes were created in wild-type and photosystem (PS) I-less backgrounds. Deletion of ndb genes led to small changes in photoautotrophic growth rates and respiratory activities. Electron transfer rates into the plastoquinone pool in thylakoids in darkness were consistent with the presence of a small amount of NDH-2 activity in thylakoids. No difference was observed between wild-type and the Ndb-less strains in the banding patterns seen on native gels when stained for either NADH or NADPH dehydrogenase activity, indicating that the Ndb proteins do not accumulate to high levels. A striking phenotype of the PS I-less background strains lacking one or more of the NDH-2s is that they were able to grow at high light intensities that were lethal to the control strain but they retained normal PS II activity. We suggest that the Ndb proteins in Synechocystis sp. strain PCC 6803 are redox sensors and that they play a regulatory role responding to the redox state of the plastoquinone pool.     

Characterization of Lysine Monomethylome and Methyltransferase in Model Cyanobacterium Synechocystis sp.PCC 6803

Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp. PCC 6803 (Synechocystis). Overall, we identified 376 monomethylation sites in 270 proteins, with numerous monomethylated proteins participating in photosynthesis and carbon metabolism. We subsequently demonstrated that CpcM, a previously identified asparagine methyltransferase in Synechocystis, could catalyze lysine monomethylation of the potential aspartate aminotransferase Sll0480 both in vivo and in vitro and regulate the enzyme activity of Sll0480. The loss of CpcM led to decreases in the maximum quantum yield in primary photosystem II (PSII) and the efficiency of energy transfer during the photosynthetic reaction in Synechocystis. We report the first lysine monomethylome in a photosynthetic organism and present a critical database for functional analyses of monomethylation in cyanobacteria. The large number of monomethylated proteins and the identification of CpcM as the lysine methyltransferase in cyanobacteria suggest that reversible methylation may influence the metabolic process and photosynthesis in both cyanobacteria and plants.     

Functional Characterization of the slr1944 Gene of Cyanobacterium Synechocystis sp. PCC 6803

The properties of Slr1944 protein encoded by the slr1944 gene and participating in the metabolism of lipophilic compounds in a cyanobacterium Synechocystis were under study. Located in the periplasm, this protein comprises a conserved pentapeptide G-X-S-X-G characteristic of lipases, acetylcholinesterases, and thioesterases. An attempt to delete the gene from the cyanobacterial genome failed; this fact presumes an essential function of Slr1944 protein under the optimum growth conditions. Expression of the slr1944 gene in Escherichia coli cells demonstrated a high affinity of the product for lipophilic compounds. An enhanced slr1944 expression deprived Synechocystis cells of the ability to restore the activity of the photosynthetic electron-transport chain following photoinactivation. The authors believe that Slr1944 participates in the biogenesis of the lipophilic components of photosynthetic complexes.     

Thylakoid Membrane Reduction Affects the Photosystem Stoichiometry in the Cyanobacterium Synechocystis sp. PCC 6803

Biogenesis of thylakoid membranes in both chloroplasts and cyanobacteria is largely not understood today. The vesicle-inducing protein in plastids 1 (Vipp1) has been suggested to be essential for thylakoid membrane formation in Arabidopsis (Arabidopsis thaliana), as well as in the cyanobacterium Synechocystis sp. PCC 6803, although its exact physiological function remains elusive so far. Here, we report that, upon depletion of Vipp1 in Synechocystis cells, the number of thylakoid layers in individual Synechocystis cells decreased, and that, in particular, the content of photosystem I (PSI) complexes was highly diminished in thylakoids. Furthermore, separation of native photosynthetic complexes indicated that PSI trimers are destabilized and the monomeric species is enriched. Therefore, depletion of thylakoid membranes specifically affects biogenesis and/or stabilization of PSI in cyanobacteria.In chloroplasts and cyanobacteria the energy transfer between PSI and PSII is regulated in a light-dependent manner (for a recent review, see Kramer et al., 2004). The two photosystems are connected by the cytochrome b₆f complex, and electron transfer from PSII via the cytochrome b₆f complex to PSI is believed to be regulated by the redox state of the plastoquinol pool potentially also involving the cytochrome b₆f complex (Fujita et al., 1987; Murakami and Fujita, 1993; Schneider et al., 2001, 2004; Pfannschmidt, 2003; Volkmer et al., 2007). Transfer of light energy to the two photosystems is mediated by light-harvesting complexes, and in cyanobacteria light is harvested by the soluble extramembranous phycobilisomes. The efficient energy transfer to PSI and PSII has to be balanced to synchronize the function of the two photosystems. In response to changing light intensities and qualities, energy coupling between the phycobilisomes and the photosystems changes, which allows a rapid adjustment of light absorbance by the individual photosystems. Furthermore, besides this short-term adaptation mechanism, it has been shown in many studies that on a longer term in cyanobacteria the ratio of the two photosystems changes depending on the light conditions (Manodori and Melis, 1986; Murakami and Fujita, 1993; Murakami et al., 1997). Upon shifting cyanobacterial cells from low-light to high-light growth conditions, the PSI-to-PSII ratio decreases due to selective suppression of the amount of functional PSI. In recent years, some genes have already been identified that are involved in this regulation of the photosystem stoichiometry (Hihara et al., 1998; Sonoike et al., 2001; Fujimori et al., 2005; Ozaki et al., 2007).Whereas in chloroplasts of higher plants and green algae the amounts of the two photosystems change in response to changing light conditions (Melis, 1984; Chow et al., 1990; Smith et al., 1990; Kim et al., 1993), it has already been noted a long time ago that the chloroplast ultrastructure also adapts to high-light and low-light conditions (Melis, 1984). Chloroplasts of plants grown under low light or far-red light have more thylakoid membranes than chloroplasts of plants grown under high light or blue light (Anderson et al., 1973; Lichtenthaler et al., 1981; Melis and Harvey, 1981). There appears to be a direct correlation between the chlorophyll content and the amount of thylakoids per chloroplast because light harvesting is increased by enhanced chlorophyll and thylakoid membrane content per chloroplast. Thus, chloroplasts adapt to high light both by a reduction of thylakoid membranes and by a decrease in the PSI-to-PSII ratio.Thylakoid membranes are exclusive features of both cyanobacteria and chloroplasts, and it still remains mysterious how formation of thylakoid membranes is organized. Many cellular processes, like lipid biosynthesis, membrane formation, protein synthesis in the cytoplasm and/or at a membrane, protein transport, protein translocation, and protein folding have to be organized and aligned for formation of internal thylakoid membranes. The recent observation that deletion of the vipp1 gene in Arabidopsis (Arabidopsis thaliana) results in complete loss of thylakoid membranes has indicated that Vipp1 is involved in biogenesis of thylakoid membranes. Further analysis has suggested that Vipp1 could be involved in vesicle trafficking between the inner envelope and the thylakoid membrane of chloroplasts (Kroll et al., 2001). Because of this, the protein was named Vipp1, for vesicle-inducing protein in plastids 1. Depletion of Vipp1 strongly affected the ability of cyanobacterial cells to form proper thylakoid membranes (Westphal et al., 2001) and, consequently, also in cyanobacteria Vipp1 appears to be involved in formation of thylakoid membranes. A Vipp1 depletion strain of Arabidopsis is deficient in photosynthesis, although the defect could not be assigned to a deficiency of a single photosynthetic complex, but appeared to be caused by dysfunction of the entire photosynthetic electron transfer chain (Kroll et al., 2001). Therefore, depletion of Vipp1 in Arabidopsis seems to affect thylakoid membrane formation rather than the assembly of thylakoid membrane protein complexes (Aseeva et al., 2007). However, for cyanobacteria, it is not clear yet how diminishing the amount of thylakoid membrane layers would affect the amount and stoichiometry of the two photosystems.Here, we present the generation and characterization of a Vipp1 depletion strain of the cyanobacterium Synechocystis sp. PCC 6803. Upon depletion of Vipp1, a decrease in thylakoid membrane pairs in the generated mutant strain and, furthermore, a significant decrease in active PSI centers was observed. Moreover, trimerization of PSI also appeared to be impaired in the mutant strain. These results suggest that thylakoid membrane perturbations caused by the Vipp1 depletion directly affects PSI assembly and stability in cyanobacterial thylakoid membranes.     

Selection by Anion-Exchange Chromatography of Exopolysaccharide Mutants of the Cyanobacterium Synechocystis Strain PCC 6803

The degree of retention of whole cells of Synechocystis strain PCC 6803 on DEAE-cellulose columns was shown to depend on their content of exopolysaccharides, which are at least in part responsible for the external negative charge of the cells. This feature was used for the isolation of mutants modified in the apparent viscosity caused by these macromolecular constituents. When a wild-type suspension was loaded onto a DE52 column, the cells eluting in the two extreme fractions of a 0 to 5 M NaCl step gradient represented 10⁻⁹ to 10⁻⁷ of the total eluted population. The accuracy of the procedure was established through the analysis of four clones: Suc(0)32 and Suc(0)65 (0 M) and Suc(5)64A and Suc(5)61 (5 M). The decreased viscosity of the exopolymers of the two 0 M clones, which appeared identical, could be related to the production of molecules less charged in uronic acids and more readily liberated from the cells. The two 5 M clones exhibited a lower sedimentation velocity, correlating with either a 60% increase in uronic acid and a doubling of the specific viscosity of the exopolysaccharides [clone Suc(5)64A] or a doubling of the per-cell production of polymers otherwise identical to those from wild-type cells [clone Suc(5)61].     

Functional Role of PilA in Iron Acquisition in the Cyanobacterium Synechocystis sp. PCC 6803

Cyanobacteria require large quantities of iron to maintain their photosynthetic machinery; however, in most environments iron is present in the form of insoluble iron oxides. Whether cyanobacteria can utilize these sources of iron, and the potential molecular mechanisms involved remains to be defined. There is increasing evidence that pili can facilitate electron donation to extracellular electron acceptors, like iron oxides in non-photosynthetic bacteria. In these organisms, the donation of electrons to iron oxides is thought to be crucial for maintaining respiration in the absence of oxygen. Our study investigates if PilA1 (major pilin protein) may also provide a mechanism to convert insoluble ferric iron into soluble ferrous iron. Growth experiments supported by spectroscopic data of a strain deficient in pilA1 indicate that the presence of the pilA1 gene enhances the ability to grow on iron oxides. These observations suggest a novel function of PilA1 in cyanobacterial iron acquisition.     

Photosynthesis and photorespiration in a mutant of the cyanobacterium Synechocystis PCC 6803 lacking carboxysomes

A mutant of the cyanobacterium Synechocystis PCC 6803 was obtained by replacing the gene of the carboxylation enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) with that of the photosynthetic bacterium Rhodospirillum rubrum. This mutant consequently lacks carboxysomes — the protein complexes in which the original enzyme is packed. It is incapable of growing at atmospheric CO₂ levels and has an apparent photosynthetic affinity for inorganic carbon (C_i) which is 1000 times lower than that of the wild type, yet it accumulates more C_i than the wild type. The mutant appears to be defective in its ability to utilize the intracellular C_i pool for photosynthesis. Unlike the carboxysomal carboxylase activity of Rubisco, which is almost insensitive to inhibition by O₂ in vitro, the soluble enzyme is competitively inhibited by O₂. The photosynthetic rate and C_i compensation point of the wild type were hardly affected by low O₂ levels. Above 100 μM O₂, however, both parameters became inhibited. The CO₂ compensation point of the mutant was linearly dependent on O₂ concentration. The higher sensitivity of the mutant to O₂ inhibition than that expected from in-vitro kinetics parameters of Rubisco, indicates a low capacity to recycle photorespiratory metabolites to Calvin-cycle intermediates.     

蓝细菌6803agp基因的分子克隆和缺失突变株的构建

PCR扩增了蓝细菌集胞藻6803(Synechocystis sp.PCC6803)的agp基因（编码ADP－葡萄糖焦磷酸羧化酶）,进一步以pUC118为载体将其克隆到大肠杆菌中,构建了pUCA质粒。通过DNA体外重组,以红霉素抗性基因部分取代agp基因片段,构建了既含agp基因上游及下游序列、又携带选择性标记－红霉素抗性的pUCAE质粒。该质粒转化野生型集胞藻6803细胞,获得了能在含红霉素的培养基上正常生长的agp基因缺失突变株。对该突变株基因组DNA进行PCR扩增,验邝了其基因结构的正确性。突变株细胞生长速度较野生型细胞快,胞内的叶绿素含量比野生型细胞高,表明该突变株具有较高的光合效率。在突变株中未检测到糖原的存在,进一步从生理水平上验证了突变株构建的正确性。     

Molecular Cloning and Construction of agp Gene Deletion-mutant in Cyanobacterium Synechocystis sp. PCC 6803

Nine compounds were isolated from Elsholtzia blanda (Benth.) Benth. Their　structures were identified with spectral and chemical methods as follows: 5,6-dihydro-6-styry-2-pyrone (1), friedelin (2), 4-hydroxy-3-methoxystyrene (3), 5,2′-dimethoxy-6,7-methylene dioxyflavanone (4), 5-hydroxy-7-methoxy-6-O-［α- L -rhamnopyranosyl(1→2)-β- D -fucopyranosyl］ flavone glycoside (5), 5,5′-dihydroxy-7-acetoxyl-6,8,3″,3″-tetramethylpyran　(3′,4′)　flavone (6), 5,5′-dihydroxy-7-(α-methyl) butyroxyl-6,8,3″,3″-tetramethylpyran (3′,4′) flavone (7), 5,5′-dihydroxy-6,7-methylenedioxy-8,3″,3″-trimethylpyran　(3′,4′)　flavone (8), glucosyringic acid (9). Among them, 6, 7 and 8 are new compounds, named as sifanghaoine Ⅰ,Ⅱ and Ⅲ, respectively.     

Systematic characterization of the ADP-ribose pyrophosphatase family in the Cyanobacterium Synechocystis sp. strain PCC 6803

We have characterized four putative ADP-ribose pyrophosphatases Sll1054, Slr0920, Slr1134, and Slr1690 in the cyanobacterium Synechocystis sp. strain PCC 6803. Each of the recombinant proteins was overexpressed in Escherichia coli and purified. Sll1054 and Slr0920 hydrolyzed ADP-ribose specifically, while Slr1134 hydrolyzed not only ADP-ribose but also NADH and flavin adenine dinucleotide. By contrast, Slr1690 showed very low activity for ADP-ribose and had four substitutions of amino acids in the Nudix motif, indicating that Slr1690 is not an active ADP-ribose pyrophosphatase. However, the quadruple mutation of Slr1690, T73G/I88E/K92E/A94G, which replaced the mutated amino acids with those conserved in the Nudix motif, resulted in a significant (6.1 x 10(2)-fold) increase in the k(cat) value. These results suggest that Slr1690 might have evolved from an active ADP-ribose pyrophosphatase. Functional and clustering analyses suggested that Sll1054 is a bacterial type, while the other three and Slr0787, which was characterized previously (Raffaelli et al., FEBS Lett. 444:222-226, 1999), are phylogenetically diverse types that originated from an archaeal Nudix protein via molecular evolutionary mechanisms, such as domain fusion and amino acid substitution.